Transposition

Evidence

Mechanisms:DNA-mediated

RNA-mediated

Transposable elements

• Mobile genetic elements - they move from one location in the genome to another

• Found in all organisms (so far studied)

• Effects:

– Insertion near or within a gene can inactivate or activate the target gene.

– Cause deletions, inversions, and translocations of DNA

– Lead to chromosome breaks

Effects of transposable elements depends on their location

Observations of B. McClintock (1930’s-1950’s)

• Certain crosses in maize resulted in large numbers of mutable loci. – The frequency of change at those loci is much higher

than normally observed.

• Studies of these plants revealed a genetic element called “Dissociation” or Ds on the short arm of chromosome 9.

• Chromosome breaks occurred at the Ds locus, which could be observed cytologically– i.e. by looking at chromosome spreads from individual

cells, e.g. sporocytes.

• Frequency and timing of these breaks is controlled by another locus, called “Activator” or Ac.

Breaks are visible cytologically on morphologically marked chromosome 9

knob

c sh bz

Wx Ds

wx

C Sh Bz

Heterochromatinbeyond knob

c sh bz

Ds

wx

c sh bz

Ds

wx

2 homologous chromosomes are distinguishable

At pachytene of meiosis, see:

OR

CEN

McClintock’s chromosome

breaks, 1952

CSHSQB

Chromosome 9, short arm, pachytene phaseof meiosis

Ds activity can appear at new locations on chromosome 9

knob Wx DsSh Bz

knob Wx

Ds

Sh Bz

knob WxDs

Sh Bz

knob WxDs

Sh Bz

I

I

I

I

Can find transpositions in the progeny

Appearance of Ds at a new location is associated with breaks: e.g. Duplications and Inversions

knob Wx DsSh BzI

Wx DsSh BzI

WxDs Sh BzI

DsWx Bz Sh inversion

WxSh BzIDs duplication

OR

In the presence of Ac,Ds events lead to variegation in sectors of kernels

I

sh bz

Wx Ds

wxC

Sh Bz

sh bz

Ds

wx

After breakage and loss of acentric chromosomes, “recessive” markers are revealed in sectors of kernels.

CEN

Ac

C

C = Colored

I>C, colorless

Variegation in sectors of kernels

Variegation in wild flox

Mechanisms of Transposition

Flanking direct repeats are generated by insertions at staggered breaks

Flanking direct repeats are generated by insertions at staggered breaks

GTTC

CAAG

5'

3'

Staggered break at the target

GTTC

CAAG

5'

3'

Insert transposable element

CAAG

5'

3'

GTTC

TE

GTTC

CAAG

FDRFDR

Transposable elements that move via DNA intermediates

• Bacterial insertion sequences– Inverted repeat at ends– Encode a transposase

• Bacterial transposons:– Inverted repeat at ends– Encode a transposase– Encode a drug resistance marker or other

marker– TnA family: transposase plus resolvase

IS elements and

transposons

Ac/Ds transposons in maize

• Ac is autonomous– Inverted repeats– Encodes a transposase

• Ds is nonautonomous– Inverted repeats– Transposase gene is defective because of

deletions in coding region

Structure of Ac and Ds

transposase

CAGGATGAAA TTTCATCCCTA

Ac

Ds

CAGGATGAAA TTTCATCCCTA

Nonfunctional transposase

deletion

Replicative vs. Nonreplicative transposition

TE

Replicon A with a transposable element = TE

Relicon B

TE

TE

TE TE

Replicative transposition:

+ +

Cointegrate

fusion of repliconsduring replicationof the TE

recombination

Nonreplicative transposition

TE

+

TE

+

Donor Recipient Donor replicon islost unless the breakis repaired.

Mechanism for DNA-mediated transposition

• Transposase nicks at ends of transposon (note cleavage is at the same sequence, since the ends are inverted repeats).

• Transposase also cuts the target to generate 5’ overhangs• The 3’ end of each strand of the transposon is ligated to the 5’

overhang of the target site, forming a crossover structure.

Crossover intermediate in transposition

Replicative transposition from the crossover structure

• The 3’ ends of each strand from the staggered break (at the target) serve as primers for repair synthesis.

• Copying through the transposon followed by ligation leads to formation of a cointegrate structure.

• Copying also generates the flanking direct repeats.• The cointegrate is resolved by recombination.

Nonreplicative transposition from the crossover structure

• Crossover structure is released by nicking at the other ends of the transposon (i.e. the ones not initially nicked).

• The gap at the target (now containing the transposon) is repaired to generate flanking direct repeats.

3-D structure of transposase and Tn5 DNA end

Transposition into a 2nd site on the same DNA molecule

Almost all transposable elements in mammals fall into one of four classes

Transposable elements that move by RNA intermediates

• Called retrotransposons• Common in eukaryotic organisms

– Some have long terminal repeats (LTRs) that regulate expression

• Yeast Ty-1• Retroviral proviruses in vertebrates

– Non-LTR retrotransposons• Mammalian LINE repeats ( long interspersed repetitive

elements, L1s)• Similar elements are found even in fungi• Mammalian SINE repeats (short interspersed repetitive

elements, e.g. human Alu repeats)• Drosophila jockey repeats• Processed genes (have lost their introns). Many are

pseudogenes.

Age distri-bution

of repeats

in human

and mouse

Mechanism of retrotransposition

• The RNA encoded by the retrotransposon is copied by reverse transcriptase into DNA

• Primer for this synthesis can be generated by endonucleolytic cleavage at the target

• Both reverse transcriptase and endonuclease are encoded by SOME (not all) retrotransposons

• The 3’ end of the DNA strand at the target that is not used for priming reverse transcriptase can be used to prime 2nd strand synthesis

Events in L1 transposition

ORF1

ORF2RT’aseendonuclease

3’ UTRpromoter

FDR FDR

transcribe

Staggered break at target

Priming of synthesis by RT’ase at staggered breakPriming of synthesis by RT’ase at staggered break

2nd strand synthesis and repair of staggered break

RT’ase works preferentially on L1 mRNA

Recombination between two nearly identical sequences (e.g transposons) will lead to

rearrangements

• Deletion if the repeats are in the same orientation

• Inversion if the repeats are in the opposite orientation

Consequences of

recombination between two transposons